The organ of Corti in the inner ear converts the mechanical force arising from sound waves into electrical signals to inform the central nervous system. To accomplish this task, hair cells and supporting cells in the organ of Corti need complex and sophisticated protein networks to perform their functions. Discovering the individual proteins and their roles in the protein networks of the organ of Corti will enrich our understanding of normal and impaired cochlear physiology. The goal of the proposed work is to determine functions of two newly identified proteins: CEACAM16 and Marshalin. CEACAM16 is a cell adhesion protein that we discovered in the cochlea. In AIM I, we propose to investigate functional roles of CEACAM16 for maintaining the tectorial membrane integrity and the connections between outer hair cells and the tectorial membrane. We will also investigate its relation to the DFNA4 deafness locus in both in vitro and in vivo environments. Marshalin is a microtubule-minus-end binding protein that we identified as a potential CDH23-associated protein. In AIM II, we will examine Marshalin expression patterns in the organ of Corti and investigate Marshalin's function in both hair cells and supporting cells during cochlear development. We'll determine the molecular basis of interactions between Marshalin and its potential associated proteins including CDH23, actin, myosin 7a and spectrin. The investigation will provide much-needed information regarding Marshalin's roles in cross-linking actin filaments and MT filaments during hair cells and supporting cells differentiation, as well as Marshalin's connection with deafness locus DFNB15, one of earliest identified deafness loci. The investigation will also offer further information regarding CDH23 role for stereocilia formation. Various cellular, biochemical and electrophysiological methods, knockout and knockin mouse models, as well as in vivo physiology methods will be used to pursue our goals. The conclusions derived from the proposed work will not only allow the possible identification of new deafness genes, but will also provide the foundation for further understanding of formation of the organ of Corti. Such knowledge is sorely needed to understand many fundamental biological questions that are directly relevant to the molecular bases of normal and disordered communication processes. PUBLIC HEALTH RELEVANCE: The goals of this proposal are to investigate two potential deafness-associated genes that play important roles in hearing. Aside from its intrinsic importance in auditory physiology, data collected from these studies will allow us to further recognize deafness-related genes and to manipulate their function for therapeutic purposes, either through molecular biological strategies, pharmacological treatments, and/or gene therapies.